This invention relates to improved Fluorescence Resonant Energy Transfer (FRET) techniques for use in high speed miniaturized assays for solutions, as well as in object-based and cell-based fluorimetry assays.
FRET provides an indication of proximity between donor and acceptor fluorophores. When a donor is excited with incident radiation at a defined frequency, some of the energy that the donor would normally emit as fluorescence is transferred to the acceptor, when the acceptor is in sufficiently close proximity to the donor (typically, within about 50 Angstroms for most donor fluorophores). At least some of the energy transferred to the acceptor is emitted as radiation at the fluorescence frequency of the acceptor. FRET is further described in various sources, such as “FRET Imaging” (Jares-Erijman, E. A, and Jovin, T. M, Nature Biotechnology, 21(11), (2003), pg 1387-1395), which is incorporated herein by reference for all purposes.
Another important concept in the context of this invention is anisotropy. Anisotropy provides a measure of the degree to which radiation is non-randomly polarized; that is, the degree to which one polarization orientation predominates over its orthogonal polarization orientation. A highly anisotropic signal will be highly polarized (for example, purely linearly polarized). A highly isotropic signal approaches random polarization. In one conventional approach, anisotropy (r) is calculated using the following equation:
  r  =                    V        ⁢                                  ⁢        V            -              g        ⁢                                  ⁢        V        ⁢                                  ⁢        H                            V        ⁢                                  ⁢        V            +              2        ⁢                                  ⁢        g        ⁢                                  ⁢        V        ⁢                                  ⁢        H            where VH and VV are the horizontal and vertical emission polarizations relative to a vertical excitation polarization and g corrects for polarization bias of the optical instrument.
Traditionally, FRET analysis relies on detecting one or more of the following: (1) the presence of fluorescence at the emission frequency of the acceptor, (2) the ratio of acceptor to donor fluorescence intensities, and (3) the lifetime of the donor's fluorescent emission. Each of these techniques has attendant difficulties. For example, merely detecting the presence of fluorescence at the emission frequency of the acceptor typically is not sufficient because the acceptor will produce some natural fluorescence when exposed to the frequency used to excite the donor fluorophore. Furthermore, time-resolved FRET imaging and analysis requires more complex instrumentation than standard fluorescence imaging and analysis.
Polarization anisotropy has been proposed as a FRET detection technique. Fluorescence generated from a FRET acceptor fluorophores is depolarized from the FRET process, and generally has relatively lower anisotropy than the fluorescence generated directly from donor fluorophores. Thus, anisotropy can be used as a measure of FRET, and, consequently, the associated proximity of donor and acceptor fluorophores. The use of this technique in homo-FRET, or FRET between like fluorophores, has been described in “Imaging molecular interactions in cells by dynamic and static fluorescence anisotropy (rFLIM and emFRET)” (Lidke, D. S., Nagy, P., Barisas, B. G., Heintzmann, R., Post, J. N., Lidke, K. A., Clayton, A. H. A., Arndt-Jovin, D. J. and Jovin, T. M., Biochem. Soc. Trans., 31(5) (2003), pg. 1020-1027), which is incorporated herein by reference for all purposes.
Fluorescence anisotropy can also be employed as a FRET detection strategy in living cells. As has been described in “High contrast imaging of fluorescent protein FRET by fluorescence polarization microscopy” (Rizzo, M. A. & Piston, D. W., Biophys J, 88 L14-16,2005), the fluorescence anisotropy for mCerulean, a type of Cyan Fluorescent Protein (CFP), has a value of about 0.3 across its entire wavelength emission range. However, when the mCerulean is in a FRET pair with mVenus, a type of Yellow Fluorescent Protein (YFP), the anisotropy remains high (slightly above 0.3) when the fluorescence is emitted by the donor (that is, the mCerulean) and decreases to about 0.15 when the fluorescence is emitted by the acceptor (that is, the mVenus). Although difference in anisotropy is very consistent between different measurements, the difference is relatively small and has limited use in experimental situations, since there are typically many unknowns and calibration factors which may affect the change in anisotropy. Thus it would be desirable to have, inter alia, an improved and more reliable method of measuring anisotropy changes associated with the FRET process.